Use of a new gene coding for a new member of the MCM2-8 family in pharmaceutical compositions

ABSTRACT

The use of the human or animal MCM9 gene, or parts of the gene, or transcripts thereof, or antisense nucleic acids able to hybridize with part of the gene or transcripts, or silencing RNA derived from parts of the transcripts and able to repress the MCM9 gene, or proteins or peptidic fragments translated from the transcripts, or antibodies directed against the proteins or peptidic fragments, for the preparation of a pharmaceutical composition for the treatment of a human or animal pathology linked to a dysfunction of the expression of the MCM9 gene, or of human or animal cancers.

The present invention relates to the use of a new gene coding for a newmember of the MCM2-8 family in pharmaceutical compositions.

The Minichromosome maintenance family (MCM2-7) comprises a group of sixstructurally related proteins required to initiate DNA synthesis ineukaryotes (Kearsey et al., 1998). These proteins function in a complexvery likely as a DNA helicase in promoting the opening of the DNA doublehelix at replication origins. ATP binding (Walker A) and hydrolysis(Walker B) motifs are present in all MCM2-7 members, embedded in aregion which is highly conserved in this protein family, also known asthe MCM2-7 signature domain (Koonin, 1993). Recently, this proteinfamily has expanded by the identification of a novel member, the MCM8protein (Gozuacik et al., 2003, Johnson et al., 2003, Maiorano et al.,2005). Unlike MCM2-7, which are widely conserved in eukaryotes, MCM8 ispresent only in higher multicellular organisms, being absent in wormsand yeast.

Very recently, another new member of the MCM protein family, the MCM9protein, has been identified in humans (Yoshida, 2005). Intriguingly,the predicted human protein (HsMCM9) has been reported to be a rathershort homolog of MCM proteins (391 aa against an average of 800 aa inMCM2-7 and MCM8 proteins) and the function of this protein is not known.

Anomalies during DNA replication process are involved in differentpathologies such as brains diseases, haematological disorders andcancers. Thus, means to control cellular division would be useful toolsfor the treatment of pathologies linked to a dysfunction of DNAreplication or for pathologies linked to an excessive cellularproliferation.

One of the aims of the invention is to provide a pharmaceuticalcomposition for treating pathologies linked to a dysfunction of DNAreplication or to an excessive cell proliferation.

One of the aims of the invention is to provide a method for inhibitingcell proliferation or enhancing DNA replication.

One of the aims of the invention is to provide a method for screeningdrugs useful in the treatment of pathologies linked to a dysfunction ofthe replication or to an excessive cell proliferation.

Another aspect of the invention relates to the identification of aMCM2-8 family protein, for which only a truncated form was known, andits specific function.

All these aspects have been obtained by the identification of thefull-length MCM9 gene and MCM9 protein.

The invention relates to the use of the human or animal MCM9 gene, orparts of said gene, or transcripts thereof, or antisense nucleic acidsable to hybridize with part of said transcripts, or silencing RNAderived from parts of said transcripts and able to repress said MCM9gene, or proteins or peptidic fragments translated from saidtranscripts, or antibodies directed against said proteins or peptidicfragments for the preparation of a pharmaceutical composition for thetreatment of a human or animal pathology linked to a dysfunction of theexpression of the MCM9 gene, or of human or animal cancers.

The Inventors have performed the complete identification of a novelmember of the MCM2-8 family represented by the members MCM2-7 and MCM8,the MCM9 protein. Like MCM8, MCM9 is only present in the genome ofhigher eukaryotes. This protein contains an MCM8-like ATP binding and-hydrolysis motif implicated in helicase activity. Strikingly, inaddition, MCM9 contains a unique carboxy-terminal domain which has onlyweak homology to MCM2-7 and MCM8, but stretches of amino acids, rangingfrom 4 to 10 amino acids, are highly conserved within MCM9 homologs. TheInventors have also shown that the very recently reported human MCM9protein, which resembles a truncated MCM-like protein missing a part ofthe MCM2-7 signature domain, is an incomplete form of the full lengthhuman MCM9 protein hMCM9 described here. Searching the human genome witheither the newly identified human MCM9 or other MCM protein sequences,The Inventors have not detected further additional members of this DNAhelicase family and suggest that it is constituted of eight members,falling into two different groups, one constituted by the MCM2-7 complexand the other by MCM8 and MCM9, which are present only in highereukaryotes.

The term “MCM2-7” refers to proteins MCM2, MCM3, MCM4, MCM5, MCM6 andMCM7.

The term “MCM2-8” refers to proteins MCM2, MCM3, MCM4, MCM5, MCM6 MCM7and MCM8.

DNA helicases have essential roles in nucleic acid metabolism,particularly during DNA replication, also called DNA duplication.Helicases are involved in unwinding DNA at replication origins, allowingDNA synthesis by recruiting DNA polymerases and they are also involvedin the whole process of the elongation and termination phases of DNAsynthesis when DNA has to be continuously and efficiently unwound. DNAhelicases bind to single strand DNA either naked or coated with thesingle strand DNA binding protein RPA (Replication Protein A) asoligomeric complexes and catalyze the melting of the DNA double helix.This reaction is catalyzed by ATP hydrolysis. ATP binds the helicase atthe ATP binding site (Walker A) and ATP hydrolysis occurs at the ATPhydrolysis motif (Walker B).

The helicase activity of a protein can be for example determined by thefollowing test: the protein to test is incubated with a single-strandedDNA substrate annealed to a 40-mer oligonucleotide for 1 hour. Thereaction products are then separated on an acrylamide gel. The helicaseactivity is revealed by the presence of single strand DNA, due to theunwinding of the dimer single-stranded DNA/oligonucleotide.

The ATPase activity can be monitored as described in Maiorano et al.(2005, Cell. 120, 315-28) or alternatively using acidic molybdate andmalachite green as follows: the protein to be tested is incubated for 10minutes at 37° C. in ATPase buffer (50 mM TrisHCl, pH 7.5; 2 mM MgCl₂;1.5 mM DTT; 0.05% Tween-20; and 0.25% μg/ml BSA) with a dT₂₅oligonucleotide or 500 ng of heat-denaturated ssDNA. The reaction isstarted by the addition of ATP and incubation at 37° C. for up to 25minutes. The reaction mixture is then transferred into themolybdate/malachite green solution and the absorbance is immediatelyread at 630 nMm (OD₃₆₀) to determine the amount of inorganic phosphateproduced during the reaction.

The expression “dysfunction of the expression of the MCM9 gene” relatesto an overexpression, a repression or an inhibition of the expression ofthe MCM9 gene, or relates to the expression of a protein coded by theMCM9 gene, which is not active or only partially active. A dysfunctionof the MCM9 gene expression can particularly induce disorders in DNAreplication.

A MCM9 protein is active or is an active form when said MCM9 protein hasan helicase activity and an ATPase activity and stimulates the formationof a pre-replication complex by loading MCM2-7 onto chromatin. Thepre-replication complex is a large protein complex made of the ORC1-6proteins, Cdc6, Cdt1 and MCM2-7 proteins.

A MCM9 protein is partially active when said MCM9 protein has anhelicase activity and/or an ATPase activity lower than the active form.

The helicase activity is lower than the helicase activity of the activeform when it represents at least 30%, particularly at least 60%, andmore particularly at least 90% of the helicase activity of the activeform.

The ATPase activity is lower than the ATPase activity of the active formwhen it represents at least 30%, particularly at least 60%, and moreparticularly at least 90% of the ATPase activity of the active form.

A MCM9 protein is not active or is an inactive form when said MCM9protein has no helicase and/or ATPase activity, or has an helicaseactivity lower than 30% of the helicase activity of the active formand/or has an ATPase activity lower than 30% of the ATPase activity ofthe active form, and/or poorly stimulates the formation of apre-replication complex by loading MCM2-7 onto chromatin.

The dysfunction of the expression of the MCM9 gene can be assayed by thedetermination of the amount of MCM9 mRNA produced in the cell either byhybridization of total cellular RNA with either a DNA or RNA probederived from the sequence of the MCM9 gene (Northern blot) or by PCRamplification of the MCM9 mRNA, following its conversion into cDNA bythe use of a Reverse Transcriptase (RT-PCR), or by in situ hybridizationwith either DNA or RNA probes derived from the sequence of the MCM9 geneafter fluorescent labelling of these probes. MCM9-specific antibodiescan be also used to determine the levels of the MCM9 protein present incells and/or tissues by western or by in situ hybridization on fixedtissues slices of isolated cells and/or nuclei.

The expression “pathologies linked to a dysfunction of the expression ofthe MCM9 gene” means that these pathologies result from disorders inhelicase activity of the MCM9 gene.

The expression “parts of said gene” means fragments of the MCM9 gene.

The invention also relates to the use of transcripts of the MCM9 gene orof parts of the MCM9 gene. The translation of these transcripts, alsocalled mRNAs, will produce the MCM9 protein, or peptidic fragments ofsaid protein. The proteins or peptidic fragments can be purified fromcells expressing said compounds. The peptidic fragments according to theinvention can also be synthesized by any method of chemistry well-knownin the art.

The invention further relates to the use of antisense nucleic acids.Antisense nucleic acids, also called antisense-oligonucleotides (AS-ONs)pair (hybridize) with their complementary mRNA target, thus blocking thetranslation of said mRNA or inducing the cleavage by RNase H of saidmRNA inside the DNA/RNA complex. In both cases, the use of antisensenucleic acids induces a specific blocking of RNA translation. Theantisense nucleic acids according to the invention comprisepreferentially 10 to 30 nucleotides. The use of antisense nucleic acidsable to hybridize with transcripts of the MCM9 gene thus allowsinhibiting the expression of the MCM9 gene.

The expression “antisense nucleic acids able to hybridize with part ofsaid transcripts” means that antisense nucleic acids will pair with partof said transcripts that are complementary under specific hybridationconditions.

Specific hybridation conditions may be determined according to“Molecular cloning”, third edition, Sambrook and Russel, CSHL press,2001.

The invention also relates to the use of silencing RNA, also calledinterfering RNA, derived from parts of transcripts of the MCM9 gene. RNAinterference is a process initiated by double-strand RNA molecules(dsRNAs), which are cut by the cell machinery into 21-23 nucleotideslong RNAs, called small interfering RNAs (siRNAs). In the cell, saidsiRNAs are then incorporated into RNA-Induced Silencing Complex (RISC),in which they guide a nuclease to degrade the target simple strand RNA.The use of silencing RNAs, which are complementary to parts of MCM9transcripts, allows the specific inhibition of the MCM9 expression.

The invention further relates to the use of MCM9 proteins or peptidicfragments of said protein, which are translated from the transcripts ofthe MCM9 gene or fragments of said gene, respectively.

The invention also relates to the use of antibodies directed againstMCM9 proteins or peptidic fragments of said protein. These antibodiesthus bind to the MCM9 protein in the cell, thus inhibiting its helicasefunction.

The invention relates in particular to the use as defined above for thepreparation of a pharmaceutical composition for the treatment ofcancers, wherein the helicase activity of MCM9 and in particular of thepolypeptide represented by SEQ ID NO 2 or SEQ ID NO 4 or SEQ ID NO 6 orSEQ ID NO 8 or SEQ ID NO 10 or SEQ ID NO 12 or SEQ ID NO 14 or SEQ ID NO16 or SEQ ID NO 18 is inactivated in tumoral cells of the human oranimal body by using silencing RNAi according to RNA interference, suchas double-stranded RNA (dsRNA) for post-transcriptional gene silencing,or short interfering RNA (siRNA) or short hairpin RNA (shRNA) to inducespecific gene suppression, or antisense DNA or RNA, or antibodies, inorder to curb the proliferation of said tumoral cells.

In a particular embodiment, the invention aims at inhibiting theproliferation of cancer cells. For that purpose, the helicase activityin tumoral cells is inactivated by specifically blocking MCM9 expressionusing RNA interference or antisense nucleotides, or by blocking the MCM9protein with specific antibodies. The level of active MCM9 andconsequently the level of helicase activity are decreased and the DNAreplication is curbed. The proliferation of the tumoral cells is thusinhibited and a stop of the DNA replication process may also induceapoptosis of the tumoral cells.

Blocking the MCM9 protein or its expression in a cell or in a specifictissue allows blocking of the cell cycle, even before DNA becomeslicensed to replicate, which is before the MCM2-7 complex is loaded ontochromatin and DNA synthesis has been initiated. Such cells, which areblocked in this early state of the cell cycle, in particular at the M toG1 phase transition, can not proceed aberrantly in the cell cycle, butwill either rest quiescent or be eliminated by apoptosis. In contrast,cells which are blocked or delayed after the licensing of their DNA forreplication or even after replication has started, have a high risk toproliferate unfaithfully, to accumulate mutations and to inherit anunstable genome. The term “unstable genome” means that said cells haverearranged the structure of their genome, e.g. by accumulation ofchromosomal abnormalities, so that they have a high probability toproliferate abnormally and to generate cancers.

Moreover, blocking MCM9 protein or its expression in a cell constitutesa more efficient treatment against cancer than current drugs that targetcells in later phases of the cell cycle, such as the S and G2 phases. Inthese phases of the cell cycle, DNA replication has already beeninitiated and therefore cells can rearrange their genome, for example byhomologous and /or non-homologous recombination, and therefore have ahigher probability to develop resistance to the said drugs. Thus, usinga drug that targets MCM9 overcomes the resistance of cancer cells to thesaid drugs, as the cell cycle is blocked before cancer cells have thepossibility to adapt their genome.

The invention particularly relates to antibodies that block the bindingof MCM9 on Cdt1 and/or on the chromatin. The inactivation of MCM9assembly with Cdt1 or with the chromatin allows blocking the entry inthe cell cycle before the loading of MCM2-7 onto chromatin, that is tosay before licensing.

The term “licensing” means giving to the chromosomes the competence toreplicate through the formation of pre-replication complexes onto DNAreplication origins.

The pre-replication complex is a large protein complex made of theORC1-6 proteins, Cdc6, Cdt1 and MCM2-7 proteins.

The protein Cdt is involved in the formation of pre-replicationcomplexes.

The helicase activity in tumoral cells is particularly inactivated byspecifically blocking the polypeptides represented by SEQ ID NO 2 or SEQID NO 4 or SEQ ID NO 6 or SEQ ID NO 8 or SEQ ID NO 10 or SEQ ID NO 12SEQ ID NO 14 or SEQ ID NO 16 or SEQ ID NO 18, or their expression.

The polypeptide represented by SEQ ID NO 2 corresponds to the human MCM9protein (1143 amino acids). The human MCM9 protein contains an ATPbinding site (Walker A) and an ATP hydrolysis motif (Walker B).

The polypeptide represented by SEQ ID NO 4 corresponds to the fragmentof the human MCM9 protein represented by SEQ ID NO 2, which extends fromamino acid 385 to 1143.

The polypeptide represented by SEQ ID NO 6 corresponds to the truncatedform of the human MCM9 protein described by Yoshida (2005), whichextends from amino acid 1 to 384 of the human MCM9 protein representedby SEQ ID NO 2.

Alignment of the truncated human MCM9 protein described by Yoshida(2005) with the other MCM proteins suggested that this protein might bea truncated MCM like protein missing the carboxy-terminal half of theMCM2-7 signature domain. Within the truncated domain, the ATP bindingsite is present but the ATP hydrolysis motif is absent. Thus, thistruncated form does not contain an ATP hydrolysis motif which isessential for DNA helicase activity (see example 1).

The polypeptide represented by SEQ ID NO 8 corresponds to the murineMCM9 protein (1290 amino acids).

The polypeptide represented by SEQ ID NO 10 corresponds to the fragmentof the murine MCM9 protein represented by SEQ ID NO 8, which extendsfrom amino acid 540 to 1290.

The polypeptide represented by SEQ ID NO 12 corresponds to the fragmentof the murine MCM9 protein represented by SEQ ID NO 8, which extendsfrom amino acid 1 to 539.

The polypeptide represented by SEQ ID NO 14 corresponds to the XenopusMCM9 protein (1143 amino acids).

The polypeptide represented by SEQ ID NO 16 corresponds to the fragmentof the Xenopus MCM9 protein represented by SEQ ID NO 14, which extendsfrom amino acid 385 to 1143.

The polypeptide represented by SEQ ID NO 18 corresponds to the fragmentof the Xenopus MCM9 protein represented by SEQ ID NO 14, which extendsfrom amino acid 1 to 384.

The efficiency of inhibition of the helicase activity, obtained byspecifically blocking MCM9 protein or its expression, can be determinedby cell proliferation test. For example, classical tests based on BrdUincorporation during DNA synthesis can be used or other tests such asanalysis of the DNA content of a cell population by FluorescenceActivated Cell Sorter (FACS), or by incorporation of either aradioactively labelled DNA precursor, or H³ (tritium) intothrichloroaceticacid (TCA) insoluble materiel, or by scoring the mitoticindex of a cell population, or by scoring the increase in the total massof a cell population (growth curve), or the increase in the rate ofprotein synthesis, or by scoring the number of Ki67-, PCNA-, MCM2-7- orCdc6-positive cells.

For the purpose of the invention, the RNA interference is obtained byusing interfering RNA chosen among double-strand RNA, short interferingRNA or short hairpin RNA. Interfering RNA can be obtained by chemicalsynthesis or by DNA-vector technology.

A short hairpin RNA is a simple strand RNA, characterized in that thetwo ends of said RNA are complementary and can hybridize together, thusforming an artificial double strand RNA with a loop between the twoends.

The invention further relates to the use as defined above for thepreparation of a pharmaceutical composition for the treatment ofneoplastic diseases such as choriocarcinoma, liver cancer induced by DNAdamaging agents or by infection by Hepatitis B virus, skin melanoticmelanoma, melanoma, premalignant actinic keratose, colon adenocarcinoma,basal cell carcinoma, squamous cell carcinoma, ocular cancer,non-Hodgkin's lymphoma, acute lymphocytic leukaemia, meningioma, softtissue sarcoma, osteosarcoma, and muscle rhabdomyosarcoma or of braindiseases such as Alzheimer disease, neuron degenerative diseases andmental retardation, or of hematological disorders.

The above-mentioned pathologies are linked to an excessive proliferationof the cells. The invention particularly relates to the use of apharmaceutical composition that allows the inhibition of theproliferation of said cells.

The invention also relates to the above-mentioned use, for thepreparation of a pharmaceutical composition for the treatment of a humanor animal pathology linked to a dysfunction of the expression of theMCM9 gene, wherein the number of functional MCM9 helicases is increasedor the activity of MCM9 helicases in cells of the human or animal bodyis stimulated by administration of functional MCM9 proteins and inparticular of polypeptides represented by SEQ ID NO 2 or SEQ ID NO 4 orSEQ ID NO 6 or SEQ ID NO 8 or SEQ ID NO 10 or SEQ ID NO 12 or SEQ ID NO14 or SEQ ID NO 16 or SEQ ID NO 18 or of fragments thereof or by gene orcell therapy.

In the above-mentioned use, the dysfunction of the expression of theMCM9 gene is linked to an inhibition or a repression of the expressionof an active form of MCM9, or to the expression of an inactive form or apartially active form of the MCM9 protein.

The above-mentioned pathologies result from the absence or the smallrate of helicase activity of the MCM9 protein, which may result from theexpression of an inactive form of the MCM9 protein or from an expressionof said protein which is between 1% to 60% smaller than the expressionin normal cells.

The administration of said pharmaceutical composition enables toincrease the number of functional MCM9 helicases or to stimulate theactivity of MCM9 helicases.

The increased number of functional helicases can be determined byimmunoblot with MCM9 specific antibodies on total cell lysates, or by insitu immunostaining on a given cell population or a tissue and/or byisolation of the MCM9 protein by immunopurification with MCM9-specificantibodies and determination of both helicase and ATPase activity invitro compared to normal cells.

The stimulation of the MCM9 helicase activity is determined byperforming an helicase test as described above in the presence of thesingle strand DNA annealed to an oligonucleotide, the single strand DNAbinding trimeric complex RPA, or with DNA polymerases, PCNA, RF-C and/orother replication fork accessory proteins.

The expression “trimeric complex” means a protein complex made of threepolypeptides.

The term “gene therapy” refers to the use of DNA as a drug. According tothe invention, said DNA comprises the MCM9 gene and is introduced in thecells so that they can express the MCM9 protein. Gene transfer methodsare well-known by the man skilled in the art. They comprise physicalmethods, such as naked DNA, microinjection, shotgun or electrotransfer,and vectorization using non-viral or viral vectors for the genetransfer.

According to the invention, the term “cell therapy” refers to the use ofcells having a normal helicase activity to replace or repair cells thatpresent a dysfunction in helicase activity.

According to another embodiment, the present invention relates to theuse as defined above for the preparation of a pharmaceutical compositionfor the treatment of pathologies characterized by a predispositiontowards cancer or premature aging and being notably caused by a defectof the helicase function and being particularly selected among Bloom'ssyndrome, Werner's syndrome, ataxia-telangectasia, xerodermiapigmentosum, Cockayne's syndrome and Rothmund-Thomson's syndrome.

The pathologies characterized by a predisposition towards cancer orpremature aging are linked to an inhibition or a repression of thehelicase activity, or to an aberrant helicase activity.

The expression “aberrant helicase activity” refers to cases wherein theDNA strand is not entirely synthesized or wherein mistakes areintroduced in the new synthesized DNA strand.

The expression “predisposition towards cancer” refers to pathologieswherein the inhibition or the repression of the helicase activity or theaberrant helicase activity can lead to aberrant new synthesized DNAstrand that may be responsible of an abnormally proliferation of thecells and then of the induction of cancers.

The expression “defect of the helicase function” refers to an inhibitionor a repression of the helicase activity and particularly leads to acell replication rate that is lower than 20%, by comparison with thereplication rate in normal (healthy) cells.

The invention relates to pharmaceutical compositions that enable torestore an helicase activity, particularly by increasing the number offunctional MCM9 helicases or to stimulating the activity of MCM9helicases.

The present invention also relates to a peptide or polypeptide of one ofthe following sequences: SEQ ID NO 2 (amino-acids 1-1143 of hMCM9), SEQID NO 4 (amino-acids 385-1143 of hMCM9), SEQ ID NO 6 (amino-acids 1-384of hMCM9), SEQ ID NO 8 (amino-acids 1-1290 of MmMCM9), SEQ ID NO 10(amino-acids 540-1290 of MmMCM9), SEQ ID NO 12 (amino-acids 1-539 ofMmMCM9), SEQ ID NO 14 (amino-acids 1-1143 of XMCM9), SEQ ID NO 16(amino-acids 385-1143 of XMCM9), SEQ ID NO 18 (amino-acids 1-384 ofXMCM9),

or derived from one of the above-defined sequences by insertion,deletion, substitution of one or more amino-acids, or flanked byadditional amino-acids at the N-terminus or at the C-terminus or at bothtermini,

provided that the resulting sequence shares at least 55%, in particularat least 65%, in particular at least 80% identity with one of theabove-defined sequences and provided that said peptide or polypeptidehas substantially the same helicase and/or ATPase activity as the MCM9protein, and in particular provided that the resulting sequence has amaximum length of 660 amino-acids and a minimum length of 300amino-acids,

or corresponding to a fragment thereof, provided that said fragment hassubstantially the same helicase and/or ATPase activity as the MCM9protein.

The expression “peptide or polypeptide derived from one of theabove-defined sequences” means that said peptide or polypeptide containsat least one mutation chosen among insertion (or addition) or deletionor substitution of one or more amino-acids, and/or that the peptide orpolypeptide is flanked by additional amino-acids at the N-terminus or atthe C-terminus or at both termini.

The mutation by substitution in the amino acid sequence can be asubstitution by a conservative amino-acid or not.

The additional amino-acids can particularly be chosen among Walker A,Walker B and Zn-finger motifs.

The derived peptides or polypeptides of the invention may be more activeforms than the native MCM9 protein and/or possess a modified helicaseand/or ATPase activity, such as being able to metabolize other forms ofATP, for example N6-benzyl ATP, N6-cyclopentyl ATP.

The expression “the resulting sequence has a maximum length of 660amino-acids and a minimum length of 300 amino-acids” particularly refersto peptides or polypeptides that are within the highly conservedMCM2-8-like N-terminus part of MCM9 protein and that comprise theMCM-2-8 family domain.

The highly conserved MCM2-8-like N-terminus part corresponds in the MCM9protein to amino acids 1-650 for Xenopus, 1-805 for Mouse and 1-650 forHuman.

The MCM-2-8 family domain corresponds in the MCM9 protein to amino acids303-606 for Xenopus, 458-761 for Mouse and 302-605 for Human.

The expression “substantially the same helicase and/or ATPase activityas the MCM9 protein” means that the helicase and/or ATPase activity ofthe derived peptide or polypeptide is at least 60%, in particular atleast 80% and more particularly at least 90% of the activity of the MCM9protein.

The present invention also relates to a peptide or polypeptide derivedfrom one of the following sequences: SEQ ID NO 2 (amino-acids 1-1143 ofhMCM9), SEQ ID NO 4 (amino-acids 385-1143 of hMCM9), SEQ ID NO 6(amino-acids 1-384 of hMCM9), SEQ ID NO 8 (amino-acids 1-1290 ofMmMCM9), SEQ ID NO 10 (amino-acids 540-1290 of MmMCM9), SEQ ID NO 12(amino-acids 1-539 of MmMCM9), SEQ ID NO 14 (amino-acids 1-1143 ofXMCM9), SEQ ID NO 16 (amino-acids 385-1143 of XMCM9), SEQ ID NO 18(amino-acids 1-384 of XMCM9),

by at least one mutation located on a site of phosphorylation by CDKs,in particular said mutations being chosen among the followings:

-   -   modification of the conserved threonine (T) in the TP motif to        alanine (A) or an equivalent amino acid and modification of the        conserved serine (S) in the SP motif to alanine (A) or an        equivalent amino acid,    -   modification of the conserved threonine (T) in the TP motif to        glutamate (E) or an equivalent amino acid and modification of        the conserved serine (S) in the SP motif to glutamate (E) or an        equivalent amino acid.

The potential SP phosphorylation sites in MCM9 proteins are located onamino acids 193-194, 478-479, 789-790, 841-842, 859-860, 965-966,1070-1071 in Xenopus, amino acids 49-50, 75-76, 94-95, 348-349, 633-634,865-866, 899-900, 910-911, 1024-1025, 1073-1074, 1204-1205, 1235-1236 inMouse, and amino acids 192-193, 477-478, 703-704, 711-712, 762-763,802-803, 883-884, 890-891, 915-916, 942-943, 952-953, 1073-1074,1088-1089 in Human.

The potential TP phosphorylation sites in MCM9 proteins are located onamino acids 370-371, 714-715, 735-736, 917-918 in Xenopus, amino acids127-128, 525-526, 1033-1034, 1054-1055 in Mouse, and amino acids369-370, 673-674, 879-880, 977-978, 1064-1065 in Human.

CDKs (Cyclin-Dependent Kinases) are enzymes involved in the regulationof cell division cycle. CDKs activate and/or inactivate their substrateby phosphorylation. CDKs recognize specific sites, called “site ofphosphorylation by CDK”, particularly the amino-acids motifs TP and SP.

The mutated forms of MCM9 proteins obtained by mutations located on asite of phosphorylation by CDKs are either inactive, partially active,active or more active than the active non mutated form.

The expression “more active than the active non mutated form” means thatthe helicase activity of the mutated form represents at least 110%,particularly at least 150% and more particularly at least 200% of thehelicase activity of the active non mutated form, and/or that the ATPaseactivity of the mutated form represents at least 110%, particularly atleast 150% and more particularly at least 200% of the ATPase activity ofthe active non mutated form.

According to the invention, the mutated forms of MCM9 are tested asdescribed above for their helicase and ATPase activity.

The invention further relates to a peptide or polypeptide derived fromone of the following sequences: SEQ ID NO 2 (amino-acids 1-1143 ofhMCM9), SEQ ID NO 4 (amino-acids 385-1143 of hMCM9), SEQ ID NO 6(amino-acids 1-384 of hMCM9), SEQ ID NO 8 (amino-acids 1-1290 ofMmMCM9), SEQ ID NO 10 (amino-acids 540-1290 of MmMCM9), SEQ ID NO 12(amino-acids 1-539 of MmMCM9), SEQ ID NO 14 (amino-acids 1-1143 ofXMCM9), SEQ ID NO 16 (amino-acids 385-1143 of XMCM9), SEQ ID NO 18(amino-acids 1-384 of XMCM9),

by at least one mutation located on a position which is essential forthe helicase and/or ATPase activity of MCM9 protein, in particular saidmutations being chosen among the followings:

-   -   modification of the conserved lysine (K) in the Walker A motif        GxxGxGK to alanine (A) or threonine (T) or other non polar or        polar neutral amino acids,    -   modification of the conserved aspartic acid (D) in the Walker B        motif DExx to alanine (A) or threonine (T) or other non polar or        polar neutral amino acids.

The expression “position which is essential for the helicase and/orATPase activity of MCM9 protein” particularly refers to Walker A andWalker B motifs.

According to the present invention, some mutated forms of the MCM9protein may lose their helicase function or have an attenuated helicaseactivity and thus may be used to decrease the proliferation of cells, inparticular of cancer cells.

The above modifications of the conserved lysine in the Walker A and/orthe conserved aspartic acid in the Walker B lead to mutated forms of theMCM9 which have a highly decreased helicase activity (less than 80% ofthe helicase activity of the wild-type MCM9 protein), or no helicaseactivity.

The mutated forms of MCM9 which have a highly decreased helicaseactivity (less than 80% of the helicase activity of the wild-type MCM9protein) or no helicase activity may be used in excess by comparison tothe native active protein, to decrease the rate of cell proliferation.

The present invention further relates to a peptide or polypeptidecontaining 65 to 160 amino-acids and comprising a fragment which isessential for the helicase function of the MCM9 protein, said fragmentcontaining in particular

sequence SEQ ID NO 20 (amino-acids 300-450 of SEQ ID NO 2) or sequenceSEQ ID NO 22 (amino-acids 310-430 of SEQ ID NO 2) or sequence SEQ ID NO24 (amino-acids 352-417 of SEQ ID NO 2) (helicase region of hMCM9)

or sequence SEQ ID NO 26 (amino-acids 460-610 of SEQ ID NO 8) orsequence SEQ ID NO 28 (amino-acids 470-590 of SEQ ID NO 8) or sequenceSEQ ID NO 30 (amino-acids 508-573 of SEQ ID NO 8) (helicase region ofMmMCM9)

or sequence SEQ ID NO 32 (amino-acids 300-450 of SEQ ID NO 14)orsequence SEQ ID NO 34 (amino-acids 310-430 of SEQ ID NO 14)or sequenceSEQ ID NO 36 (amino-acids 353-418 of SEQ ID NO 14) (helicase region ofXMCM9)

or sequence SEQ ID NO 38 (amino-acids 352-359 of SEQ ID NO 2) (walker Amotif of hMCM9)

or sequence SEQ ID NO 40 (amino-acids 414-417 of SEQ ID NO 2) (walker Bmotif of hMCM9)

or sequence SEQ ID NO 42 (amino-acids 508-515 of SEQ ID NO 8) (walker Amotif of MmMCM9)

or sequence SEQ ID NO 44 (amino-acids 570-573 of SEQ ID NO 8) (walker Bmotif of MmMCM9)

or sequence SEQ ID NO 46 (amino-acids 353-360 of SEQ ID NO 14) (walker Amotif of XMCM9)

or sequence SEQ ID NO 48 (amino-acids 415-418 of SEQ ID NO 14) (walker Bmotif of XMCM9),

or any fragment derived therefrom by insertion, deletion, substitutionof one or more amino acid or sharing at least 50%, in particular atleast 65%, in particular at least 80% identity therewith, provided thatthe resulting fragment substantially retains at least part of thehelicase and/or ATPase activity of the MCM9 protein.

The expression “fragment which is essential for the helicase function ofMCM9 protein” refers to a fragment that particularly comprises orconsists of the Walker A motif and/or the Walker B motif.

Walker A motif is involved in ATP binding. This motif forms aGlycin-rich flexible loop preceded by a β-strand and followed by anα-helix. The Walker A motif of Xenopus and mammalian MCM9 homologs(Gozuacik et al., 2003; Johnson et al., 2003) is a canonical consensussequence (GxxGxGKS/T).

Walker B motif is involved in ATP hydrolysis and has the followingstructure: hybrophobic stretch followed by the amino acids signatureD[ED], where the presence of at least one negatively charged amino acidin this motif is crucial for its function.

The expression “helicase region” refers to a region of the MCM9 proteinthat possesses the helicase activity, particularly by comprising theWalker A motif and/or the Walker B motif.

The invention also relates to a nucleic acid encoding the peptides orpolypeptides as defined above.

The term “nucleic acid” refers to DNA or RNA.

The invention relates to single stranded or double stranded nucleicacids.

The invention further relates to a nucleic acid of one of the followingsequences: SEQ ID NO 1 (nucleotides 1-4798 of hMCM9), SEQ ID NO 3(nucleotides 1153-4798 of hMCM9), SEQ ID NO 5 (nucleotides 1-1152 ofhMCM9), SEQ ID NO 7 (nucleotides 1-3873 of MmMCM9), SEQ ID NO 9(nucleotides 1618-3873 of MmMCM9), SEQ ID NO 11 (nucleotides 1-1617 ofMmMCM9), SEQ ID NO 13 (nucleotides 1-3432 of XMCM9), SEQ ID NO 15(nucleotides 1153-3432 of XMCM9), SEQ ID NO 17 (nucleotides 1-1152 ofXMCM9),

or derived from one of the above-defined sequences by insertion,deletion, substitution of one or more nucleotide, or flanked byadditional nucleotides at the 5′ end or 3′ end or at both ends,

provided that the resulting sequence shares at least 40%, in particularat least 60%, in particular at least 80% identity with one of theabove-defined sequences and provided that the resulting sequence encodesa peptide or polypeptide which has substantially the same helicaseand/or ATPase activity as the MCM9 protein, and in particular providedthat the resulting sequence has a maximum length of 1980 nucleotides anda minimum length of 900 nucleotides,

or corresponding to a fragment thereof, provided that said fragmentencodes a peptide or polypeptide which has substantially the samehelicase and/or ATPase activity as the MCM9 protein.

The nucleotide sequence represented by SEQ ID NO 1 (nucleotides 1-4798)encodes the human MCM9 helicase represented by SEQ ID NO 1.

The nucleotide sequence represented by SEQ ID NO 3 corresponds to thenucleotides 1153-4798 of the human MCM9 sequence represented by SEQ IDNO 1 and encodes the polypeptide of sequence SEQ ID NO 4.

The nucleotide sequence represented by SEQ ID NO 5 corresponds to thenucleotides 1-1152 of the human MCM9 sequence represented by SEQ ID NO 1and encodes the polypeptide of sequence SEQ ID NO 6.

The nucleotide sequence represented by SEQ ID NO 7 (nucleotides 1-3873)encodes the murine MCM9 helicase represented by SEQ ID NO 8.

The nucleotide sequence represented by SEQ ID NO 9 corresponds to thenucleotides 1618-3873 of the murine MCM9 sequence represented by SEQ IDNO 7 and encodes the polypeptide of sequence SEQ ID NO 10.

The nucleotide sequence represented by SEQ ID NO 11 corresponds to thenucleotides 1-1617 of the murine MCM9 sequence represented by SEQ ID NO7 and encodes the polypeptide of sequence SEQ ID NO 12.

The nucleotide sequence represented by SEQ ID NO 13 (nucleotides 1-3432)encodes the Xenopus MCM9 helicase represented by SEQ ID NO 14.

The nucleotide sequence represented by SEQ ID NO 15 corresponds to thenucleotides 1153-3432 of the Xenopus MCM9 sequence represented by SEQ IDNO 13 and encodes the polypeptide of sequence SEQ ID NO 16.

The nucleotide sequence represented by SEQ ID NO 17 corresponds to thenucleotides 1-1152 of the Xenopus MCM9 sequence represented by SEQ ID NO13 and encodes the polypeptide of sequence SEQ ID NO 18.

The expression “nucleic acid derived from one of the above-definedsequences” means that said nucleic acid contains at least one mutationchosen among insertion (or addition) or deletion or substitution of oneor more nucleotide, and/or that the nucleic acid is flanked byadditional nucleotides at the 5′ end or at the 3′ end or at both ends.

The mutation by deletion or by addition in the nucleic acid caneventually induce a shift in the opening reading frame of the MCM9nucleotide sequence, in a way that the peptide or polypeptide encoded bysaid nucleic acid has substantially the same function as the MCM9protein.

The mutation by substitution in the nucleotide sequence can lead to asilencing substitution due to the degeneracy of the genetic code, or toa substitution by a conservative amino-acid or a non conservativeamino-acid in the peptide or polypeptide encoded by said nucleotideacid.

The additional nucleotides can particularly be chosen among nucleotidesthat encode Walker A, Walker B and Zn-finger motifs.

The expression “the resulting sequence has a maximum length of 1980nucleotides and a minimum length of 900 nucleotides” particularly refersto nucleotide sequences encoding peptides or polypeptides that arewithin the highly conserved MCM2-8-like N-terminus part of MCM9 proteinand that comprise the MCM-2-8 family domain.

The invention also relates to a nucleic acid derived from one of thefollowing sequences: SEQ ID NO 1 (nucleotides 1-4798 of hMCM9), SEQ IDNO 3 (nucleotides 1153-4798 of hMCM9), SEQ ID NO 5 (nucleotides 1-1152of hMCM9), SEQ ID NO 7 (nucleotides 1-3873 of MmMCM9), SEQ ID NO 9(nucleotides 1618-3873 of MmMCM9), SEQ ID NO 11 (nucleotides 1-1617 ofMmMCM9), SEQ ID NO 13 (nucleotides 1-3432 of XMCM9), SEQ ID NO 15(nucleotides 1153-3432 of XMCM9), SEQ ID NO 17 (nucleotides 1-1152 ofXMCM9),

by at least one mutation, the resulting sequence encoding a peptide or apolypeptide having at least a mutation located on a site ofphosphorylation by CDKs, in particular chosen among the followings:

-   -   modification of the conserved threonine (T) in the TP motif to        alanine (A) or an equivalent amino acid and modification of the        conserved serine (S) in the SP motif to alanine (A) or an        equivalent amino acid,    -   modification of the conserved threonine (T) in the TP motif to        glutamate (E) or an equivalent amino acid and modification of        the conserved serine (S) in the SP motif to glutamate (E) or an        equivalent amino acid.

The invention further relates to a nucleic acid derived from one of thefollowing sequences: SEQ ID NO 1 (nucleotides 1-4798 of hMCM9), SEQ IDNO 3 (nucleotides 1153-4798 of hMCM9), SEQ ID NO 5 (nucleotides 1-1152of hMCM9), SEQ ID NO 7 (nucleotides 1-3873 of MmMCM9), SEQ ID NO 9(nucleotides 1618-3873 of MmMCM9), SEQ ID NO 11 (nucleotides 1-1617 ofMmMCM9), SEQ ID NO 13 (nucleotides 1-3432 of XMCM9), SEQ ID NO 15(nucleotides 1153-3432 of XMCM9), SEQ ID NO 17 (nucleotides 1-1152 ofXMCM9),

by at least one mutation, the resulting sequence encoding a peptide or apolypeptide having at least a mutation located on a position which isessential for the helicase and/or ATPase activity of MCM9 protein, inparticular said mutations being chosen among the followings:

-   -   modification of the conserved lysine (K) in the Walker A motif        GxxGxGK to alanine (A) or threonine (T) or other non polar or        polar neutral amino acids,    -   modification of the conserved aspartic acid (D) in the Walker B        motif DExx to alanine (A) or threonine (T) or other non polar or        polar neutral amino acids.

The invention further relates to a nucleic acid which contains 180 to480 nucleotides and which comprises a fragment which encodes a part ofthe MCM9 protein which is essential for its helicase function, saidfragment containing in particular

sequence SEQ ID NO 19 (nucleotides 898-1350 of SEQ ID NO 1) or sequenceSEQ ID NO 21 (nucleotides 928-1290 of SEQ ID NO 1) or sequence SEQ ID NO23 (nucleotides 1054-1251 of SEQ ID NO 1) (helicase region of hMCM9)

or sequence SEQ ID NO 25 (nucleotides 1387-1830 of SEQ ID NO 7)orsequence SEQ ID NO 27 (nucleotides 1408-1770 of SEQ ID NO 7) or sequenceSEQ ID NO 29 (nucleotides 1522-1719 of SEQ ID NO 7) (helicase region ofMmMCM9)

or sequence SEQ ID NO 31 (nucleotides 898-1350 of SEQ ID NO 13) orsequence SEQ ID NO 33 (nucleotides 928-1290 of SEQ ID NO 13) or sequenceSEQ ID NO 35 (nucleotides 1057-1254 of SEQ ID NO 13) (helicase region ofXMCM9)

or sequence SEQ ID NO 37 (nucleotides 1054-1077 of SEQ ID NO 1) (walkerA motif of hMCM9)

or sequence SEQ ID NO 39 (nucleotides 1240-1251 of SEQ ID NO 1) (walkerB motif of hMCM9)

or sequence SEQ ID NO 41 (nucleotides 1522-1545 of SEQ ID NO 7) (walkerA motif of MmMCM9)

or sequence SEQ ID NO 43 (nucleotides 1708-1719 of SEQ ID NO 7) (walkerB motif of MmMCM9)

or sequence SEQ ID NO 45 (nucleotides 1057-1080 of SEQ ID NO 13) (walkerA motif of XMCM9)

or sequence SEQ ID NO 47 (nucleotides 1243-1254 of SEQ ID NO 13) (walkerB motif of XMCM9)

or any fragment derived therefrom by insertion, deletion, substitutionof one or more nucleotide or sharing at least 45%, in particular atleast 60%, in particular at least 80% identity therewith, provided thatthe resulting fragment encodes a peptide or polypeptide whichsubstantially retains at least part of the helicase and/or ATPaseactivity of the MCM9 protein.

The invention relates to a nucleic acid which is complementary to anucleic acid as defined above.

The term “complementary” means that said nucleic acid is able to pair orhybridize to a nucleic acid by Watson and Crick or other base-pairinteractions, thus being able to form a double-stranded structure withthis nucleic acid.

The invention also relates to a nucleic acid which is capable ofhybridizing with a nucleic acid as defined above under appropriatehybridizing conditions.

The “appropriate hybridizing conditions” may be determined according to“Molecular cloning”, third edition, Sambrook and Russel, CSHL press,2001.

The invention described herein also relates to an expression vectorcomprising a nucleic acid as described above and the elements which arenecessary for its expression in a cell.

The expression “elements which are necessary for its expression”particularly refers to regulatory sequences to which the nuclei acid isoperably linked.

The term “operably linked” means that the nucleotide sequence is linkedto a regulatory sequence in a manner which allows the expression of thenucleic acid sequence. The regulatory sequences are well known by theman skilled in the art. They include promoters, enhancers and otherexpression control elements.

The invention also provides a cell transformed by a nucleic acid asdefined above or by an expression vector as defined above.

The host cell according to the present invention includes prokaryotichost cells (bacterial cells), such as E. coli, Streptomyces,Pseudomonas, Serratia marcescens and salmonella typhimurium oreukaryotic cells such as insect cells, in particularbaculovirus-infected Sft9 cells, or fungal cells, such as yeast cells,or plant cells or mammalian cells.

The invention further relates to a recombinant protein obtained by theexpression of the expression vector as defined above.

The DNA vector containing the MCM9 gene or fragments thereof as definedabove is used to produce a recombinant form of the protein byrecombinant technology. Recombinant technology comprises the steps ofligating the nucleotide sequence into a gene construct such as anexpression vector and transforming or transfecting said gene constructinto host cells. The host cells that express the protein are then lysedand the recombinant protein is isolated and purified, for example bychromatography.

The present invention relates to an antibody or antigen-binding fragmentwhich binds to an MCM9 protein or part of an MCM9 protein or to amodified active MCM9 protein, in particular to a peptide or polypeptideas defined above and in particular to the polypeptide represented by SEQID NO 2 or SEQ ID NO 4 (corresponding to amino-acids 1-1143 or 385-1143of hMCM9).

The antibody can be polyclonal or monoclonal and the term “antibody” isintended to encompass both polyclonal and monoclonal antibodies. Theterms “polyclonal” and “monoclonal” refer to the degree of homogeneityof an antibody preparation, and are not intended to be limited to aparticular method of production.

The present invention relates to antibodies which bind to MCM9 proteinor part of an MCM9 protein, or to a mutated form of the MCM9 protein orpart thereof. A mammal, such as a rabbit, a mouse or a hamster, can beimmunized with an immunogenic form of the protein, such as the entireprotein or a part of it. The protein or part of it can be administeredin the presence of an adjuvant.

The term “immunogenic” refers to the ability of a molecule to elicit anantibody response. Techniques for conferring immunogenicity to a proteinor part of it which is not itself immunogenic include conjugation tocarriers or other techniques well known in the art.

The immunization process can be monitored by detection of antibodytiters in plasma or serum. Standard immunoassays, such as ELISA can beused with the immunogenic protein or peptide as antigen to assess thelevels of antibody.

The invention relates in particular to a monoclonal or polyclonalantibody directed against an MCM9 protein or against a peptide orpolypeptide comprising part of an MCM9 protein, and in particularagainst a peptide or polypeptide as defined above.

The invention also relates to a method for the in vitro or ex vivoproduction of catalytically active MCM9 helicase in foreign expressionsystems,

such as bacteria (E. coli) or insect cells (Sf9), or equivalent or invitro systems for coupled transcription/translation of the MCM9 cDNA,such as rabbit reticulocytes systems or lysate of E. coli cells ortranslation of the MCM9 mRNA into xenopus oocyte or egg extracts,

possibly under form of a tagged recombinant protein, comprising thesteps of:

-   -   lysis of cells expressing MCM9 proteins in the following buffer        or equivalent, 20 mM TrisHCl pH 8.5, 100 mM KCl, 5 mM        β-mercaptoethanol, 5-10 mM imidazole, 10% glycerol (v/v)        proteases inhibitors;    -   purification of the soluble MCM9 proteins by nickel affinity        chromatography technology or equivalent or similar affinity        chromatography technology;    -   elution of bound proteins in 10 mM TrisHCl pH 8.5; 100 mM KCl; 5        mM β-mercaptoethanol; 100-250 mM imidazole, 10% glycerol (v/v)        proteases inhibitors;    -   supplementation of purified MCM9 proteins, with or without        cleaved tag, with 0.1 mg/ml of BSA;    -   desaltation on a Bio-spin P30 column (Biorad) equilibrated with        20 mM TrisHCl pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 0.01%        Triton X-100 for helicase and ATPase activities, or in XB (100        mM KCl, 0.1 mM CaCl₂, 2 mM MgCl₂, 10 mM Hepes-KOH, 50 mM        sucrose, pH 7.7) for egg extracts reconstitution experiments;        and    -   supplementation of the protein with 25% glycerol and storage at        −20° C. mercaptoethanol; The rabbit reticulocytes systems and        lysate of E. coli cells are ex vivo cell free extracts that can        transcribe a given cDNA into mRNA and translate the mRNA into a        protein. Such a system may be valuable to produce catalytically        active protein to perform in vitro activity assays.

The recombinant proteins are tagged either at the N- or C-terminal withwell-known sequence Tag, such as Hist-Tag, Myc-Tag, Flag-Tag, Tap-Tag,GST-tag, MAL-Tag, in order to facilitate the purification of theprotein. Preferentially, the sequence tag can be removed by an enzymaticor chemical reaction involving the use of thrombin and/or TEV proteaseor similar enzymatic activities.

The expression “catalytically active” means that the correspondingrecombinant protein can bind and hydrolyze continuously ATP resulting inhelicase activity, such as displacement of an oligonucleotide annealedto single stranded DNA, or able to melt double stranded DNA in vitro,and/or that the protein can catalyze formation of pre-replicationcomplexes in vitro and /or in vivo, determined by the ability of MCM2-7proteins to associate with chromatin in vivo and /or in vitro.

According to another embodiment, the invention relates to apharmaceutical composition comprising as active substance a peptide orpolypeptide or a nucleic acid or an expression vector or a cell or anantibody or antigen-binding or a monoclonal or polyclonal antibody, asdefined above, in association with a pharmaceutically acceptablevehicle.

The pharmaceutical preparation of the present invention can beformulated with a physiologically acceptable medium, such as water,buffered saline, polyols (glycerol, propylene glycol, liquidpolyethylene glycol) or dextrose solutions. Preferentially, thepharmaceutical preparation is formulated in a vector which will allowthe delivery of said preparation inside the target cells. Thepharmaceutical preparation can be administered by intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous or oral way.

The pharmaceutical preparation may also be administered as part of acombinatorial therapy with other agents, such as inhibitors oractivators of cell proliferation. Inhibitors of cell proliferation canbe chosen among aphidicoline, cis-platinum, etoposides, lovastatin,mimosine, nocodazole. Activators of cell proliferation can be chosenamong growth factors such as EGF (Epidermal Growth Factor), FGF(Fibroblast Growth Factor), NGF (Nerve Growth Factor) and analogues, andlipopolysaccharides.

The invention relates to a method for the screening of drugs useful inthe treatment of human or animal pathology linked to a dysfunction ofthe expression of the MCM9 gene, said method comprising contacting ofthe potential drugs with cells such as cancer cells or transformed cellsand especially liver, brain, muscle, skin or gut cells wherein adecrease of the expression of the MCM9 helicase is induced bytransformation of said cells with recombinant and/or mutated forms ofthe MCM9 gene which is in particular represented by one of the followingsequences: SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, or of partsof said gene, or of transcripts thereof, or of antisense nucleic acidsable to hybridize with part of said gene or transcripts, or of silencingRNA derived from parts of said transcripts and able to repress said MCM9gene, and screening the drugs able to inhibit the proliferation of saidtransformed cells.

According to another embodiment, the present invention relates to amethod for the in vitro or ex vivo screening of drugs useful in thetreatment of human or animal pathology linked to a dysfunction of theexpression of the MCM9 gene, said method comprising contacting of thepotential drugs with cells such as:

-   -   cancer cells or    -   cells wherein recombinant and/or mutated active forms of MCM9        helicase, which is in particular represented by one of the        following sequences: SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ        ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16,        SEQ ID NO 18, or fragments thereof, are introduced to increase        the helicase activity in said cells or    -   transformed cells and especially liver, brain, muscle, skin or        gut cells wherein an increase of the expression of an active        form of MCM9 helicase is induced by transformation of said cells        with recombinant and/or mutated forms of the MCM9 gene which is        in particular represented by one of the following sequences: SEQ        ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ        ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, or of parts        of said gene, or of transcripts thereof,        and screening the drugs able to inhibit the proliferation of        said cells.

The expression “to increase the helicase activity” means that thehelicase activity is increased from 10% to 90%, in particular from 50%to 90%, by comparison with the basal helicase activity of wild-type MCM9protein.

The expression “basal helicase activity” refers to the helicase activityin cells cultured in usual conditions, particularly according to themanufacturer protocol.

The expression “increase of the expression of an active form of MCM9helicase” means that the number of active helicase is increased, thusincreasing the helicase activity as defined above.

In the above embodiment, the term “drugs” refers to inhibitors of DNAreplication whose target is the DNA helicase. The inhibitors of DNAreplication can be chosen among dibenzothiepin and its analogues,non-hydrolysable NTPs such as γATP, DNA-interacting ligands such asnogalamycin, daunorubicin, ethidium bromide, mitoxantrone, actinomycin,netropsin and cisplatin, 4,5,6,7-tetrabromo-1H-benzotriazole (TBBT),peptides binding DNA that inhibit the unwinding of the double helix bythe helicase, bananins and its derivatives, theaminothiazolylphenyl-containing compounds BILS 179 BS and BILS 45 BS,5’-O-(4-fluorosulphonylbenzoyl)-esters of ribavirin (FSBR), adenosine(FSBA), guanosine (FSBG) and inosine (FSBI), CDKs inhibitors such asstaurosporines and its derivatives.

In order to screen potential drugs inhibiting cell proliferation,proliferation tests are carried out on the proliferative cells.

According to another embodiment, the present invention relates to amethod for the in vitro or ex vivo screening of drugs useful in thetreatment of human or animal pathology linked to a dysfunction of theexpression of the MCM9 gene, said method comprising contacting of thepotential drugs with:

-   -   cells wherein recombinant and/or mutated inactive forms of MCM9        helicase are introduced to decrease the helicase activity in        said cells or    -   transformed cells and especially liver, brain, muscle, skin or        gut cells wherein an increase of the expression of an inactive        MCM9 helicase is induced by transformation of said cells with        recombinant and/or mutated forms of the MCM9 gene which is in        particular represented by one of the following sequences: SEQ ID        NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID        NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, or of parts of        said gene, or of transcripts thereof, or,    -   transformed cells and especially liver, brain, muscle, skin or        gut cells wherein a decrease of the expression of an active form        of MCM9 helicase is induced by transformation of said cells with        antisense nucleic acids able to hybridize with part of said gene        or transcripts, or of silencing RNA derived from parts of said        transcripts and able to repress said MCM9 gene, or    -   cells wherein antibodies directed against MCM9 protein, which is        in particular represented by one of the following sequences: SEQ        ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10,        SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, or        fragments thereof, are introduced to decrease the helicase        activity in said cells, and        screening the drugs able to stimulate the proliferation of said        cells.

The expression “to decrease the helicase activity” means that thehelicase activity is decreased from 10% to 90%, particularly from 30% to90%, more particularly from 60% to 90%, by comparison with the basalhelicase activity of wild-type MCM9 protein.

The expression “decrease of the expression of an active form of MCM9helicase” means that the number of active helicase is decreased, thusdecreasing the helicase activity as defined above.

In the above embodiment, the term “drugs” refers to activators of DNAreplication whose target is the DNA helicase. The activators of DNAreplication can be chosen among caffeine, tamoxifen in uterine tissues,leptomycin B, CDKs inhibitors such as staurosporines.

In order to screen potential drugs stimulating cell proliferation,proliferation tests are carried out on the non proliferative cells.

The invention also relates to the use of an agonist or antagonist of anMCM9 helicase and in particular of the polypeptide represented by SEQ IDNO 2 or SEQ ID NO 4 or SEQ ID NO 6 or SEQ ID NO 8 or SEQ ID NO 10 or SEQID NO 12 or SEQ ID NO 14 or SEQ ID NO 16 or SEQ ID NO 18, for inhibitingcell proliferation or allowing to increase replication of the DNA,particularly in vitro or ex vivo, wherein the agonist or antagonistenters the cell, said antagonist causing the inhibition of DNAreplication and said agonist contributing to the restoration of cellproliferation or to the ability of the cell to replicate DNA inunfavorable conditions.

The expression “restoration of cell proliferation” means that theproliferation of the cells, for example blocked by the action of anantagonist, can be re-established, so that cells can replicate the DNAand divide.

The expression “unfavorable conditions” refers to conditions whereincells are not competent to proliferate, because they are in a state ofquiescence, such as differentiated cells, or because the proliferationof said cells has been temporarily blocked by an inhibitor of cellproliferation, such as mimosine, lovastatine, aphidicolin, hydroxyurea,or DNA damaging agents and/or alkylating agents.

The invention particularly relates to antagonists of MCM9 that block thebinding of MCM9 on cdt1 and/or on the chromatin.

The invention further relates to a method for inhibiting cellproliferation or allowing a better replication of the DNA, comprisingadministering an agonist or antagonist of an MCM9 helicase and inparticular of the polypeptide represented by SEQ ID NO 2 or SEQ ID NO 4or SEQ ID NO 6 or SEQ ID NO 8 or SEQ ID NO 10 or SEQ ID NO 12 or SEQ IDNO 14 or SEQ ID NO 16 or SEQ ID NO 18 in a way that the agonist orantagonist enters the cell, said antagonist causing the inhibition ofDNA replication and said agonist contributing to the restoration of cellreplication or to the ability of the cell to replicate DNA inunfavorable conditions.

The invention also relates to a method for inhibiting cell proliferationor allowing a better replication of the DNA in vitro or ex vivo,comprising administering an agonist or antagonist of an MCM9 helicaseand in particular of the polypeptide represented by SEQ ID NO 2 or SEQID NO 4 or SEQ ID NO 6 or SEQ ID NO 8 or SEQ ID NO 10 or SEQ ID NO 12 orSEQ ID NO 14 or SEQ ID NO 16 or SEQ ID NO 18 in a way that the agonistor antagonist enters the cell, said antagonist causing the inhibition ofDNA replication and said agonist contributing to the restoration of cellreplication or to the ability of the cell to replicate DNA inunfavorable conditions.

The invention also relates to a method for diagnosing, in particular invitro or ex vivo, a pathology or a risk of developing a pathology linkedto a disorder in the expression of the MCM9 protein, consisting inassessing

-   -   a possible surexpression of MCM9 or    -   a possible alteration of the normal activity of MCM9 and/or    -   a possible mutation on a MCM9 gene and/or    -   a possible mutation on a MCM9 protein in particular resulting in        a possible genomic instability and/or    -   a possible neoplastic transformation.

The expression “genomic instability” refers to the loss and/oralteration of the genetic material during cell proliferation anddivision.

The surexpression of MCM9 is assessed by measuring the level ofexpression of the MCM9 gene by Northern blot and /or RT-PCR in vivoand/or in vitro, or by in situ hybridization of cells with DNA and/orRNA probes, as well as by determining the amount of MCM9 proteinproduced in the cell by western blot and/or by in situ hybridizationwith MCM9-specific antibody (immunofluorescence). The levels ofexpression of the MCM9 gene and/or of the corresponding protein in cellssurexpressing MCM9 are compared to the levels of non-pathologic cellsisolated from the same patient.

The alteration of the normal activity of MCM9 is assessed by determiningthe DNA helicase activity of the MCM9 protein in vitro and/or in vivo,and/or by assessing the ability of MCM9 to catalyze the formation ofpre-replication complexes onto chromatin in vivo and/or in vitro. Thislatter can be determined by detection of components of thepre-replication complex, such as the MCM2-7 proteins, and/or that of thePCNA protein, onto chromatin by western blot and/or immunofluorescencein vivo and/or in vitro.

The mutation on a MCM9 gene is assessed by extraction and isolation ofthe DNA from the cells and determination of the DNA sequence of the MCM9gene, and/or by isolation of the total mRNAs from the cells andamplification of the MCM9 gene by RT-PCR, and/or by analysis of thepolymorphysm of the MCM9 gene by restriction digest (RFLP).

The mutation on a MCM9 protein in particular resulting in a possiblegenomic instability is assessed by comparison of the sequence of theMCM9 gene isolated from pathologic cells with that isolated fromnon-pathologic cells obtained from the same patient. Mutations in theDNA sequence coding for the known motifs of the MCM9 protein, such asthe Zn-finger domain and/or the MCM2-8 signature domain and/or thehelicase domain are potential candidates for mutations causing genomicinstability.

The neoplastic transformation is assessed by the ability of cells toproliferate indefinitely in vitro and/or the ability of said cells toinduce tumors when injected into animals.

The invention also relates to a method for the screening of biologicallyactive agents useful in the treatment of human or animal pathologylinked to a dysfunction of the expression of the MCM9 gene, said methodcomprising:

-   -   administering a potential agent to a non-human transgenic animal        model for MCM9 gene function, particularly chosen among a MCM9        knock-out model and a model of exogenous and stably transmitted        MCM9 sequence, and    -   determining the effect of said agent on the development of the        transgenic animal and/or the development of diseases such as        those defined above, and in particular the development of        cancer.

The term “non-human animal” includes all mammals expect for humans,advantageously rodents and in particular mice.

The term “transgenic animal” denotes an animal into whose genome hasbeen introduced an exogenous gene construct, which has been insertedeither randomly into a chromosome, or very specifically at the locus ofan endogenous gene.

In a MCM9 knock-out model, the exogenous gene construct has beeninserted at the locus of the MCM9 gene, resulting in the impossibilityof expressing this MCM9 gene, since it is either interrupted or entirelyor partially replaced by a construct such that it no longer allowsexpression of the endogenous gene, or alternatively a construct which,in addition to the deletion of the endogenous gene, introduces anexogenous gene. Such animals will be referred to as “knock-out” animalsor animals in which the abovementioned endogenous gene is invalidated.

A model of exogenous and stably transmitted MCM9 sequence can beobtained by transfection of the cells of the animal (such as stem cellsor in vitro cultured cell lines) with a DNA plasmid bearing wild-type ormutated forms of the MCM9 gene under control of promoter sequence of theMCM9 gene or promoters for standard reporter genes which areconstitutively expressed or whose expression can be controlled byinduction with inducers of the expression of the above mentionedpromoters, integration of such plasmid in the chromosome of such cellsso that this transgene is stably transmitted to the cell progeny.

The effect of the agent is determined by morphological and/orphenotypical analysis of the transgenic animal, and/or by molecularanalysis by measure of cell proliferation and/or cell death and/or celldifferentiation and/or cell apoptosis, and/or determination of thekaryotype of the animal, that is to say analysis of the number andstructure of the chromosomes of cells chosen from the whole embryo ortissues of the animal.

DESCRIPTION OF THE FIGURES

FIG. 1A and FIG. 1B

The MCM9 protein is a novel member of the MCM2-8 protein family with anunique C-terminal domain.

FIG. 1A: Alignment of the previously reported truncated human MCM9protein truncated hMCM9 (Yoshida, 2005) with the Xenopus MCM9 protein(XlMCM9). The MCM2-8 signature domain is shown in grey. The ATP binding(Walker A) and hydrolysis (Walker B) motifs are indicated. Numbersindicate aminoacids.

FIG. 1B: Alignment between full length MCM9 homologs in differentorganisms: Xenopus (XlMCM9), chicken (GgMCM9), mouse (MmMCM9) and human(hMCM9) proteins. Numbers indicate aminoacids.

FIG. 2A, FIG. 2B and FIG. 2C

Alignment of MCM9-like proteins in different organisms.

FIG. 2A: Alignment of the conserved N-terminal half of MCM9 proteinsfrom Humans (SEQ ID NO: 49), Mouse (SEQ ID NO: 50), Chicken (SEQ ID NO:51) and Xenopus (SEQ ID NO: 52), obtained by ClustalW. The Walker A andB motifs are underlined. Stars indicate identity, while similar aminoacids are indicated by a single or double dot. The number of pointsindicates the degree of similarity defined by the program ClustalW.

FIG. 2B: Alignment of MCM9 (SEQ ID NO: 60) with MCM2-8 proteins (MCM3(SEQ ID NO: 53), MCM7 (SEQ ID NO: 54), MCM5 (SEQ ID NO: 55), MCM2 (SEQID NO: 56), MCM4 (SEQ ID NO: 57), MCM6 (SEQ ID NO: 58) and MCM8 (SEQ IDNO: 59)) from Xenopus within the central region of MCM2-8 proteins andthe N-terminal of MCM9. Alignment was performed as in FIG. 2A. Walker Aand B motifs are underlined. The alignment region corresponds to aminoacids 449-673 for MCM2, 285-520 for MCM3, 446-673 for MCM4, 320-545 forMCM5, 335-563 for MCM6, 321-550 for MCM7, 336-650 for MCM8 and 291-550for MCM9.

FIG. 2C: Holograms of human MCM2-7, MCM8 and MCM9. The Phylogram wascalculated with ClustalW.

FIG. 2D: Alignment of the C-terminal regions of MCM9 from Xenopus (SEQID NO: 61), Chicken (SEQ ID NO: 62), Mouse (SEQ ID NO: 63) and Humans(SEQ ID NO: 64). Alignment was performed as in FIG. 2A. The alignmentregion corresponds to amino acids 650-1143 for Xenopus MCM9, 800-1291for Mouse MCM9, 650-1169 for Chicken MCM9 and 650-1143 for Human MCM9.

FIG. 3

Characterization of the antibody raised against a peptide correspondingto amino acids 605 to 1143 of the Xenopus MCM9 protein.

Western Blot analysis of egg extract (1 μl and 2 μl) with pre-immuneserum (lane 1 and 2) and serum against MCM9 (3IP1, lane 3 and 4).

FIG. 4

MCM9 associates with chromatin.

Western blot of chromatin fractions prepared after 40, 60 or 120 minutesafter addition of sperm chromatin to egg extract. MCM9 and ORC2 proteinswere detected with specific antibodies. The last lane (40+geminin) showsthat MCM9 associates with chromatin also when replication is blocked bygeminin, however to a lesser extent.

FIGS. 5A and 5B

Depletion (Δ) of MCM9 from egg extracts abolishes DNA replication.

FIG. 5A: Western blot of egg extract after incubation with non-specificantibody (Mock) or antibody against MCM9. The MCM9 protein can becompletely removed from the egg extract as seen when depletion iscarried out using an antibody against MCM9 (Δ MCM9).

FIG. 5B: Replication kinetics in Mock-depleted egg extract (squares) andMCM9-depleted egg extract (circles). Depletion of MCM9 abolishes DNAreplication.

FIG. 6

Depletion of MCM9 from an egg extract does not (quantitatively)codeplete other proteins involved in DNA licensing/replication.

Egg extract after Mock-depletion (A Mock) or MCM9-depletion (Δ MCM9) wasmixed with SDS-sample buffer, loaded on a SDS-PAGE and blotted.Following western blot analysis was performed for MCM9, as well as forproteins known to be involved in DNA licensing or replication (allMCM2-7, MCM8, Cdt1 and ORC2).

FIG. 7

Depletion of MCM9 from egg extract inhibits the loading of the MCM2-7complex onto chromatin.

A western blot analysis of chromatin assembled either in a Mock-depleted(Δ Mock) or MCM9-depleted (Δ MCM9) egg extract, was carried out asdescribed in FIG. 4. The presence of MCM2-7, Cdt1, CDC6 and ORC2 in thechromatin was detected using the corresponding antibodies.

Chromatin which was assembled in MCM9-depleted extract does not containMCM2-7 proteins, less Cdt1, but more CDC6.

FIG. 8

Association of MCM9 with chromatin is dependent on ORC.

A western blot analysis of chromatin assembled either in a Mock-depleted(Δ Mock) or ORC2-depleted (Δ ORC2) egg extract was carried out as inFIG. 4. Chromatin which was assembled in an ORC2-depleted extract doesnot contain MCM9 and is also devoid of pre-RC proteins (MCM2-7, Cdt1,CDC6). Histon H3 (H3) is shown as a loading control and ORC2 as adepletion control.

FIG. 9

MCM9 interacts with Cdt1 in egg extract.

Western blot analysis of immunopurifications (IP) of MCM9, Cdt1, and aMock-purification (done with an unspecific antibody), using an MCM9antibody (upper lane) or a cdt1 antibody (lower lane). In the MCM9purification, a substantial amount of Cdt1 is present, showing thatthese two proteins interact in egg extract.

FIG. 10

GST-TEV-MCM9 can be purified from Baculovirus-infected SF9 cells.

Coomasie stained SDS-PAGE showing GST-TEV-MCM9 recombinant protein boundto GSH-beads, after incubation of the GSH-beads with SF9 cell-lysate,and the GST-TEV-MCM9 protein after elution from the GSH-beads (eluateGSH).

FIGS. 11A and 11B

RT-PCR analysis of human MCM9 RNA.

FIG. 11A: RT-PCR analysis of parts of human MCM9 (fragments A, B, C andD)

FIG. 11B: location of the fragments amplified by PCR in FIG. 11A on thehuman MCM9 cDNA.

EXAMPLES Example 1 Identification of MCM9 Protein, a Specific VertebrateMember of the MCM8 Protein Family

Screening the public EST databases, the inventors have identified ahomolog of the truncated human MCM9 protein in Xenopus laevis. Unlikethe reported truncated human MCM9, the Xenopus MCM9 (XlMCM9) is muchlonger and contains all the features of MCM proteins, in particular theentire MCM2-7 signature domain, made of both ATP binding (Walker A) andhydrolysis (Walker B) motifs. In addition, Xenopus MCM9 is closerrelated to MCM8, since both possess the canonical Walker A and B motifs(whereas MCM2-7 possesses a deviant Walker A motif).

By careful screening the genome of other vertebrates and mammals insilico, the Inventors have now identified conserved homologs of theentire MCM9 protein also in chicken, mouse and human, whose primarystructure closely resembles that of the Xenopus MCM9 protein. Thesefindings indicate that the MCM9 protein is a canonical MCM protein alsoin humans, closer related to human MCM8 than to human MCM2-7 proteins,and that the previously reported truncated human MCM9 protein representsonly a part of the entire human MCM9.

Experimental Procedures

Identification of MCM9 Homologs

To identify homologs of MCM9, database searches were performed using theprogram BLAST. Either EST databases or genomic databases for indicatedorganisms were searched. In addition, ab initio proteins were generatedto identify hypothetical proteins by BLAST with the GNOMON routine.GNOMON uses multiple heuristics to find the best self-consistent set oftranscripts and protein alignments in a certain genomic region. Theprogram calculates splice sites and identifies the cases where two exonsof the protein alignment are as closed as maximum 50 by having differentframes. Since such short introns are extremely rare, in these casesGNOMON introduces frame shifts in the sequence to combine multipleexons, which allows to create consistent transcripts also from genomicregions containing errors in its sequence. Proteins predicted by GNOMONwere then confirmed by identification of EST sequences in EST databasesand by GEO Blast.

Special Search for Short Regions of High Homology

To identify smaller regions of homology and to identify EST sequenceswithin a database, also MEGABLAST was used (Zhang et al., 2000) which isespecially suited for the identification of shorter, but highly similarsequences in a given genome database. Megablast was designed to optimizethe alignment of sequences which differ only slightly due to e.g.sequencing errors.

Protein Alignment

Protein alignments were performed using the program ALIGN (Pearson etal., 1997) or CLUSTALW (Higgins et al., 1994), available on the serverof the Institute of Human Genetics (IGH), Montpellier or the EMBL-EBIserver. Identification of protein domains and motif searches wereperformed either using InterProScan available on the EMBL-EBI serverscanning the InterPro database of protein families, domains andfunctional sites (Mulder et al., 2005) or MotifScan, using theHits-database from the Swiss Institute of Bioinformatics (SIB).

Results

Identification of a Xenopus Homolog of Reported Truncated Human MCM9

To identify a Xenopus homolog of the recently described truncated humanMCM9 protein (Yoshida, 2005), the Inventors performed a search using theBLAST program with the truncated hMCM9 protein sequence as a queryagainst the Expressed Sequences Tagged (EST) Xenopus database.Consequently, the Inventors identified the cDNA clone IMAGE6637819(accession number BC070720 on GenBank), coding for a protein of 1143amino acids derived from a mRNA expressed in Xenopus eggs. Sequencealignment with XlMCM proteins show that the first 835 aa of XlMCM9 share25.6% identity with full length XlMCM8 (835 aa) while the identity withXlMCM2-7 proteins is in average 10.5%. These results strongly indicatethat XlMCM9 is a distinct member of the MCM family in Xenopus. (FIG.1A). XlMCM9 shows a strong identity (73.8%) in its first amino-terminal391 aa with the reported truncated hMCM9.protein (391 aa, Yoshida,2005). However, unlike the reported truncated hMCM9, the XlMCM9 proteincontains a much longer carboxy-terminal extension which shows in itsfirst part a high homology to the other MCM proteins. Within thisregion, XlMCM9 contains an intact MCM2-7 family signature domain (aa303-aa 606) harboring Walker A and B motifs. The MCM2-7 family domain isthe highest conserved region among the members of the MCM2-7 family.

Interestingly, the Walker A motif of XlMCM9 (GxxGxGKS, aa 354-360), is acanonical consensus site as the one found in MCM8 proteins, butdifferent from that found in MCM2-7 proteins, which is a deviantconsensus site (GxxGxAK/S). The Inventors conclude that this protein isthe Xenopus homologue of truncated hMCM9. Importantly, the size of theXlMCM9 protein is bigger than that of other MCM proteins. This isessentially due to a C-terminal extension after the MCM homology region,which does not share a clear homology to other MCM proteins and seems tobe a unique feature of this protein (FIG. 1 A).

Identification of MCM9 Homologs in the Genome of Other Vertebrates andMammals

Given that the length of XlMCM9 is much bigger than reported for thetruncated hMCM9 (Yoshida, 2005), the Inventors investigated whether thiswas a special feature of the Xenopus protein or if a longer MCM9 homologprotein could be also identified in other organisms. Therefore, theInventors performed databank searches using BLAST with the XlMCM9protein sequence against databases of several organisms.

A record (XM_(—)419764 on GenBank) in the chicken genomic database wasfound derived from an annotated sequence (NW_(—)060336 on GenBank,located on chromosome 3 between 61.196 and 61.290 kbp). Within thisregion, GNOMON predicts a mRNA coding for a 1169 aa long protein, whichcould be supported by multiple EST evidences (e.g. BU378776, BU478046,BU271359, on GenBank). This chicken MCM9 (GgMCM9) shares 54.1% identitywith XlMCM9. Like XlMCM9, the chicken protein consists of two mainparts: an N-terminal part which is highly conserved (aa 1-626 share81.2% identity with XlMCM9) and a C-terminal region which is much lessconserved within MCM proteins as well as in respect to other MCM9homologs (FIG. 2D).

Next, searching the mouse genome database, the Inventors found twoentries (BAB31238.1 and NP_(—)954598 on chromosome 10 between 53.544 and53.679 kbp, on GenBank), both coding for unnamed protein products. Theproteins corresponding to these sequences showed 87% identity with theN-terminus of GgMCM9, and 47.9% identity with the carboxy-terminus ofGgMCM9, respectively. Searching the mouse EST database with thesesequences, a number of partially overlapping expressed sequences wereidentified (e.g. BY720667, CB244669, CX2225903, on GenBank) and theentire corresponding protein was re-joined in silico, resulting in a1291 aa long hypothetical protein possessing over 60% identity with theGgMCM9 protein and 47% to XlMCM9. MmMCM9 shares the general organizationof a highly conserved N-terminus and a much less conserved C-terminus inrespect to the other identified MCM9 proteins and other MCM familymembers (FIG. 1B). In addition, the first 150 aa of this protein are notpresent in the other MCM9 proteins. Importantly, its first 386 aa are100% identical with the reported 386 aa containing mouse MCM9 (Yoshida,2005).

These findings suggest that XlMCM9-like proteins can also be found inthe genome of other vertebrates and mammals. Therefore, the Inventorsre-investigated the human databases using the full XlMCM9 sequence as aquery to search for a complete human MCM9 protein. First, homologs ofXlMCM9 were searched in the human genome with BLAST. Two overlappingsequence entries on chromosome 6 were found (NT_(—)025741 andNT_(—)086697, on GenBank) revealing highest alignment significance. Theidentified human sequences were coding for amino acid stretches, whichwere highly similar to XlMCM9 over the entire length of the protein,strongly suggesting that a human MCM9 with a similar size as the Xenopusprotein exists.

Next, using the GNOMON routine within BLAST (which corrects artificialframe shifts, see Materials and Methods 2.1), to generate ab initioproteins, a human MCM9 was found at exactly the same position onchromosome 6, highly similar to XlMCM9. This protein was in itsN-terminus 100% identical to the first 385 aa of the reported truncated391 aa long hMCM9 (Yoshida, 2005). Consequently, multiple partiallyoverlapping EST sequences corresponding to the human MCM9 region werealso identified (e.g. CV030253, CX756843 (which contains the full WalkerA and B motif), DR008069, on GenBank), demonstrating that a mRNA of theprotein inclusive an intact Walker B motif and an elongated C-terminusis indeed transcribed.

Finally the Inventors searched by BLAST the human genome with the hMCM9protein generated by GNOMON. Over 30 BLAST hits on chromosome 6 werefound, covering nearly all the hMCM9 sequence generated by GNOMON,giving direct EST evidence from aa 1 to aa 1060. Some hits were locatedat the locus previously annotated as MCMDC1 (as MCM-containing domain 1)at the position 6q 22.31, corresponding to the 7 exons of truncatedhMCM9 previously described (Yoshida, 2005). In addition, more hits wereidentified further downstream of the MCMDC1 locus and beyond the ASF1gene, which is located in an intron of HMCM9 and transcribed in theopposite direction, corresponding to 6 more exons of the hMCM9 gene.Finally, on the map of the human chromosome 6 at position 6q22.31, theentire open reading frame of the HsMCM9 gene with the correspondingprotein is also annotated as the entry hmm17631 in the GNOMON model inMap viewer, as member of the MCM2/3/5 family. Thus, this new hMCM9 geneconsists of 13 exons, giving rise to a mRNA of 4789 nucleotidescontaining 1366 nucleotides of untranslated 3′ sequences. Thecorresponding hMCM9 protein consists of 1143 aa, thus having a similarlength as the identified proteins in Xenopus, mouse and chicken. Theseresults show that the recently reported truncated hMCM9 (Yoshida, 2005)is an N-terminal fragment of the whole protein and that the stop-codonreported in this sequence and considered as the end of the protein,corresponds to the end of exon seven.

The here identified full length hMCM9 shares 55.0% identity with XlMCM9and 63.8% identity with the MmMCM9 over its entire length (FIG. 2A and2B). These findings show that all new identified members of the MCM9family (Xenopus, chicken, mouse and human) are similar in length andhighly conserved.

Characterization and Classification of the New MCM9 Proteins

The most striking feature of the MCM9 protein in different organisms istheir highly conserved N-terminus (aa 1-650), which contains allclassical features of MCM2-7 and MCM8 proteins, including Zn finger-likedomains, the Walker A and B motifs as well as a full MCM2-7 familydomain (FIG. 1A and FIG. 2A). Only the mouse protein appears to containadditional 150 aa on its N-terminus. However, MCM9 shares a much higherhomology to MCM8 than to the other MCM2-7 proteins (FIG. 2A and FIG. 2C)and it is only present in vertebrates. Thus, MCM8 and MCM9 represent adistinct sub-family of MCM DNA helicases, perhaps to fulfill specialneeds which came up with the more complex biology and development ofmulticellular organisms, especially in vertebrates. Indeed, MCM8 andMCM9 are present in vertebrates, but are absent in yeast, worms andflies. In contrast, the C-terminal half of all identified MCM9 proteins(aa 650 to the end), is less conserved (FIG. 2B), unique and not presentin other MCM proteins, although a weak homology to human MCM8 exists. Noobvious protein signatures or motifs could be identified with asignificant score within this part. However, the C-terminus containsseveral short, nevertheless highly conserved stretches. The elongatedC-terminus of this newly identified MCM9 protein might not be directlyinvolved in helicase activity, but in binding to other factors orhelicases.

Conclusions

The newly identified MCM9 protein seems to be generally present invertebrates (e.g. also in dog within the contig NW_(—)139836, onGenBank), cow (XP_(—)584574, on protein sequence database) and zebrafish (within the contig CAAK01001524.1, on GenBank) whereas in D.melanogaster, C. elegans and S. cerevisiae there appears to be no MCM9homolog. The previously identified HsMCM9 protein, which is shorter insize than MCM proteins, was annotated as MCMDC1 in the GenBank publicdatabase (NM_(—)153255), suggesting that this protein may be a proteinfunctionally unrelated to MCM proteins, but sharing some homology withthem, in particular in one part of the MCM2-7 signature. These findingsclarify this issue by establishing that MCM9 is a canonical MCM protein,more related to MCM8 than to the six MCM2-7 proteins and whose motifsand sequences are conserved in vertebrates and mammals, includinghumans.

Example 2 MCM9, a Protein Essentially Involved in Pre-RC Formation andInitiation of DNA Replication

Experimental Procedures

Plasmid Constructs

The following vectors containing Xenopus MCM9 or parts of the gene weremade:

-   -   for expression in E. coli: pET24d-MCM9 (EcoRI/SalI, aa1-1143),        pProEXHTGST-TEV-MCM9 (EcoRI/SalI, aa1-1143), pProEXHT-Strict1        (EcoRI/XhoI, aa605-779), pProEXHT-C-term (EcoRI/XhoI, aa        605-1143), and    -   for expression using Baculovirus: pFastBacGST-TEV-MCM9        (EcoRI/SalI, aa1-1143), pFastBacHT-MCM9 (EcoRI/SalI, aa1-1143),        pFastBacGST-TEV-NMCM9 (EcoRI/EcoRI, aa1-654), pFastBacHT-NMCM9        (EcoRI/EcoRI, aa1-654).

The PET plasmids were obtained from Novagen and Invitrogen.

Cloning was performed using standard PCR techniques. As template toamplify the ORF of XlMCM9 cDNA clone with the Image ID 6643889 (onPubmed) was used. Via PCR, restriction sites (see above for eachconstruct) were introduced before and after the ORF. The vector and thePCR product were then digested with the indicated restriction enzymesand ligated using T4 ligase. Ligations were tranformed into E. coliDH5α, clones were grown, plasmids purified and analyzed for successfulligation using restriction digests.

Antibodies

Polyclonal antibodies against two different recombinant parts of theXenopus laevis MCM9 (aa605-779) and (aa605-1143) were raised in rabbits.

Rabbits were injected with around 1 mg of protein mixed with Freud'sadjuvant (1:1) in intervals of 3 weeks. The first bleed (3IP1) was taken12 days after the third injection and was used for western blotting anddepletion.

In intervals of several weeks, antigen was re-injected and more serumcollected, which equally was used for western bloting and depletion.

Both antibodies recognize the protein in Xenopus egg extracts. Forexample, the Western blot in FIG. 3 shows that the antibody raisedagainst the amino acids 605 to 1143 of MCM9 recognizes the MCM9 proteinin Xenopus egg extracts.

Polyclonal antibodies against the human hMCM9 protein were raised inrabbits as described above, using the following peptides forimmunization:

human peptide 1 (aa 989-1008): (SEQ ID NO: 65) ETKEVSQQPPEKHGPREKVM, andhuman peptide 2 (aa 809-828): (SEQ ID NO: 66) PWRADNVESNKKKRLALDSE.

Antibodies against ORC-2 were obtained from Dr. J. Walter, HarvardUniversity, Boston, USA. Antibodies against CDC6 were described inLemaître et al., 2002, Nature. Antibodies against all MCM2-7 weredescribed in Maiorano et al., 2000, JBC, antibodies against MCM8 inMaiorano et al., 2005, Cell, and antibodies against cdt1 in Maiorano etal., 2000, Nature.

The Mock antibody is obtained from rabbit serum before immunization withthe MCM9 recombinant protein.

RT-PCR

Extraction of total human RNA from HeLa cells was performed usingstandard techniques with the RNeasy Mini Kit (Qiagen, Cat. Nr. 74104).Purified RNA (1 ug) was reversed transcribed using SuperScript IIIFirst-Strand Synthesis System (Invitrogen, Cat. Nr. 18080-051) with poly(dT) primers. Next, PCR reaction was performed using Pfu Turbo DNAPolymerase (Stratagene, Cat. Nr. 600250) with specific primers forhMCM9.

To obtain the fragments of human MCM9 shown in FIG. 11, the followingprimers were used:

for fragment A: (SEQ ID NO: 67) 5′: TAC AGG AAC ACG GGT CAG(SEQ ID NO: 68) 3′: GAA ACA TCA GGC GAG CAT for fragment B:(SEQ ID NO: 69) 5′: TAC AGG AAC ACG GGT CAG (SEQ ID NO: 70)3′: TGC CAT GAA ATC AAA CCA ATC for fragment C (SEQ ID NO: 71)5′: TTT GAT TTC ATG GCA ACT CAT (SEQ ID NO: 72)3′: CGC ATT GGA GCT GTG GTT GTA for fragment D: (SEQ ID NO: 73)5′: TTG ATA GTG CAC TGC GAA GGT (SEQ ID NO: 74)3′: TGC ATT ACA ATC CCG TAA A

Cloning of the entire cDNA of human MCM9 was performed using thefollowing primers:

(SEQ ID NO: 75) 5′ GGGGGGGTCGACCAGCCATTACCTAGATTCAAG 3′ (forward)(SEQ ID NO: 76) 5′ GGGGGGCTCGAGCAGAAAGCTTTTCCCAACTA 3′ (reverse).Proteins Expression and Purification

For expression in E. coli, vectors were transformed into E. coli codonplus strain (Stratagene) and cultivated in minimal medium. Expressioncultures were grown at 37 degrees to an OD of 0.3, then shifted to roomtemperature and at an OD of 0.8 induced for two hours with 0.5 mM IPTG.Cells were harvested by centrifugation and frozen in liquid N2.

For expression in SF9 cells, a construct was transformed in DH10bac E.coli (Kit Invitrogen), colonies containing recombinant virus DNA wereidentified and the DNA purified and transfected into SF9 cells. Aftervirus amplification, 500 ml cultures of infected SF9 were grown andfrozen in liquid N2.

To purify recombinant GST (Glutathione S-transferase)-MCM9 from Sf9cells, cells were lysed in Dicis-Buffer (300 mM NaCl, 150 mM KoAC, 20 mMTris pH 6.8, 2 mM MgCl2, 10% Glycerol, 0.01 NP40)+0.1% NP40.) bysonication. After centrifugation (15 min, 15 000 rpm in an SS34 rotor),the supernatant was incubated either with Glutathione-Sepharose 4B(Amersham Bioscience) or Ni-NTA Sepharose (Qiagen) for 40 min at 4° C.After binding, the GSH (glutathione)-beads were washed with 20 volumesof Dicis-Buffer. Elution of the GST-fusion protein was performed usingone volume of Dicis-buffer+20 mM GSH at RT for 15 min for theGlutathione-Sepharose beads. Ni-NTA beads were washed withDicis-buffer+20 mM imidazol and eluted stepwise with Dicis-buffersupplemented with 50, 100 and finally 150 mM.

To purify recombinant His-MCM9, the protocol is similar to the aboveprotocol of GST-MCM9 purification, except that the protein is bound toNi-beads (resins) and is eluted with increasing concentrations ofimidazole in the buffer (50, 100, 150 mM imidazole).

Xenopus Egg Extracts and DNA Replication Reactions

Egg extracts, were prepared and used as previously described (Mechaliand Harland, 1982; Menut et al., 1988). Depletion and reconstitutionexperiments were as previously described (Maiorano et al., 2000b).

Briefly, Xenopus low speed egg extracts were supplemented withcycloheximide (250 μg/ml) and double-depleted with anti-MCM9 serumcoupled to Protein-A sepharose beads or recombinant protein A sepharose(Pharmacia, 50% beads to extract ratio), for 40 minutes at 4° C.

Xenopus Egg Extracts

Egg extracts were prepared as described previously (Menut et al, 1998).Upon thawing, egg extracts were supplemented with cycloheximide (250μg/ml) and an energy regeneration system (10 μg/ml creatine kinase; 10mM creatine phosphate; 1 mM ATP; 1 mM MgCl₂). To follow DNA replicationby incorporation of α-[³²P] dNTP into newly replicated DNA, 1 μl ofα-[³²P] dNTP (3000 Ci/mmol) was added to standard reaction of 50 μl.

Immunopurification Procedures

Immunopurification was performed by incubation of egg extract with theindicated antibody (MCM9, Cdt1 or unspecific antibody) for 60 min at 4degrees. Then, Protein A-sepharose was added and incubated for another60 min at 4 degrees. Next, the Protein A-sepharose was extensivelywashed with XB and bound proteins were finally eluted with SDS-samplebuffer. Samples were separated on a SDS-PAGE and analyzed by westernblotting.

Chromatin Purification

Sperm DNA was incubated in egg extract for the 40, 60 or 120 minutes.Chromatin fractions were obtained by diluting with 5 volumes of XBbuffer (10 mM HEPES-KOH pH 7.7, 100 mM KCl, 0.1 mM CaCl 2, 1 mM MgCl2,5% sucrose)+0.3% Triton X-100, keeping them for 5 min at 4° C. Next, theextraction was purified by centrifugation through a sucrose cushion (0.7M Sucrose in XB). The pellet containing the chromatin fraction wassolubilized in sample buffer for SDS-PAGE analysis.

Results:

Biological Characterization of MCM9

In FIG. 4, chromatin purifications after indicated times were blottedagainst MCM9 and ORC2 (as a loading control). Lanes 3 and 4 show thatMCM9 associates with chromatin. Lane 2 is a negative control (no DNAadded). The last lane (+Geminin) shows that MCM9 is also present onchromatin when replication is blocked by the addition of geminin.However, MCM9 is likely to be less stable than other proteins involvedin DNA replication like ORC, CDC6 or Cdt1.

FIG. 5B shows that depletion of MCM9 from an egg extract abolishes DNAreplication. First, the complete depletion of MCM9 was assessed by awestern blot of egg extract, after incubation with non-specific antibody(Mock) or antibody against MCM9. The results shown in FIG. 5A indicatethat the MCM9 protein can be completely removed from the egg extract asseen in Δ MCM9. Then, a replication kinetics of Mock-depleted eggextract (squares) and MCM9-depleted egg extract (circles) was carriedout. The results shown in FIG. 5B indicate that depletion of MCM9abolishes DNA replication.

A western blot analysis of egg extract either Mock-depleted orMCM9-depleted for other proteins involved in DNA licensing orreplication was then carried out. The results shown in FIG. 6 indicatethat the depletion of MCM9 from an egg extract does not (quantitatively)codeplete other proteins involved in DNA licensing/replication. Thus,MCM9 is essential for DNA replication and, by depleting only MCM9, DNAreplication is abolished.

FIG. 7 shows a western blot analysis of chromatin assembled either in aMock-depleted or MCM9-depleted egg extract. Chromatin which wasassembled in MCM9-depleted extract does not contain MCM2-7 proteins andless Cdt1 than in Mock-depleted egg extract. Thus, depletion of MCM9from egg extract inhibits the loading of the MCM2-7 complex onchromatin. This result indicates that blocking MCM9 protein allowsstopping DNA replication at an early stage, before the production ofsingle strand DNAs.

FIG. 8 shows a western blot analysis of chromatin assembled either in aMock-depleted or ORC2-depleted egg extract. Chromatin which wasassembled in an ORC2-depleted extract is devoid of pre-RC proteins(Cdt1, CDC6, MCM2-7) and also does not contain MCM9. Histon H3 (H3) isshown as a loading control. Association of MCM9 with chromatin is thusdependent on ORC (Origin Recognition Complex). MCM9 certainly bindschromatin at the replication origin via ORC.

In FIG. 9, MCM9 is shown to interact with Cdt1 in egg extract: a westernblot analysis of immune purifications of MCM9, Cdt1 and aMock-purification (done with an unspecific antibody) was carried out. Inthe MCM9 purification, a substantial amount of Cdt1 is present, showingthat these two proteins interact in egg extract (FIG. 9). Thus, MCM9binds chromatin by associating with cdt1, a protein which is necessaryfor replication.

Recombinant Expression of MCM9

GST-TEV-MCM9 is purified from Baculovirus-infected SF9 cells. In FIG.10, Coomasie stained SDS-PAGE shows GSH-beads loaded with GST-TEV-MCM9after incubation with SF9 cell-lysate and the GSH-eluated proteinGST-TEV-MCM9.

Evidence for the MCM9 in Human Cells

An RT-PCR analysis was performed on human cDNA with specific primerspairs corresponding to different fragments of the human MCM9. Thesefragments represented in FIG. 11B overlap on almost the full-lengthhuman MCM9 protein. The results shown in FIG. 11A reveal the presence ofspecific bands corresponding to the different parts of the human MCM9.

References

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The invention claimed is:
 1. An isolated polypeptide consisting of oneof the following sequences: SEQ ID NO: 2, and SEQ ID NO:
 8. 2. A methodfor the in vitro or ex vivo production of catalytically active MCM9helicase in foreign expression systems, or in vitro systems for coupledtranscription/translation of the MCM9 cDNA, or translation of the MCM9mRNA into xenopus oocyte or egg extracts, comprising the steps of: a)lysing of cells expressing MCM9 proteins in the following buffer orequivalent, 20 mM TrisHCl pH 8.5, 100 mM KCl, 5 mM β-mercaptoethanol,5-10 mM imidazole, 10% glycerol (v/v), proteases inhibitors; b)purifying the soluble MCM9 proteins by nickel affinity chromatographytechnology or equivalent or similar affinity chromatography technology;c) eluting bound proteins in 10 mM TrisHCl pH 8.5, 100 mM KCl, 5 mMβ-mercaptoethanol, 100-250 mM imidazole, 10% glycerol (v/v), proteasesinhibitors: d) supplementing purified MCM9 proteins, with 0.1 mg/ml ofBSA; e) desalting on a Bio-spin P30 column (Biorad) equilibrated with 20mM TrisHCl pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 0.01% TritonX-100 for helicase and ATPase activities, or in XB (100 mM KCl, 0.1 mMCaCl₂, 2 mM MgCl₂, 10 mM Hepes-KOH, 50 mM sucrose, pH 7.7) for eggextracts reconstitution experiments; and f) supplementing the purifiedMCM9 proteins with 25% glycerol and storage at −20° C; wherein the MCM9protein is the isolated polypeptide according to claim
 1. 3. Apharmaceutical composition comprising the isolated polypeptide of claim1 as an active substance, and a pharmaceutically acceptable vehicle.